1. Technical Field
The present invention generally relates to cell transplantation and specifically to methods of improving cell viability, graft survival, the viability of cryopreserved cells and providing increased numbers of differentiated cells for transplant.
2. Background Art
Transplantation of cells and tissues is being utilized therapeutically in a wide range of disorders from cystic fibrosis (lungs), kidney failure, degenerative heart diseases to neurodegenerative disorders. Improved means to facilitate such transplants are needed, particularly where differentiated cells are being transplanted. Generally these cells cannot be cultured to increase cell number so preservation of viability for transplant is critical. Further, the number of differentiated cells available for transplant is often low and methods of increasing the number and/or availability of such cells are also needed.
Transplantation protocols in addition to transplanting tissues and/or organs can include the infusion of cell suspensions from a donor. A wide range of transplantable material either currently being transplanted or contemplated for transplants includes skin grafts, corneas, hepatic tissue, kidneys, hearts, islet cells, neurons, bone, bone marrow, and the like. The obligatory step for the success of this kind of treatment is to have enough viable cells or organs a available for the transplant.
For example, in some cancer therapies a patient's bone marrow is removed, and then reinfused following high dose chemo- and/or radiation therapies. It would be useful to have improved methods of preserving such autologous cells. In other cases, bone marrow must be from donors. Often there is not a match from relatives and the marrow must be matched from the national registry. It would be useful to be able to have preserved such cells rather than having to find the donor at the time the cells are needed. Therefore, improved methods of cryopreservation are needed since a substantial portion of cryopreserved cells are not viable upon thawing.
In addition, hybridomas are stored utilizing cryopreservation. Improved methods for preserving hybridomas with increased viability would be useful.
As a further example, the central nervous system (CNS) (brain and spinal cord) has poor regenerative capacity which is exemplified in a number of neurodegenerative disorders. An example of such a disorder is Parkinson's disease. The preferred pharmacotherapy for Parkinson's disease is L-dopa which helps the symptoms of this disease in humans. However, the neuropathological damage and the debilitating progression is not reversed by this pharmaceutical treatment protocol.
Laboratory and clinical studies have shown the transplantation of cells into the CNS is a potentially significant alternative therapeutic modality for neurodegenerative disorders such as Parkinson's disease (Wictorin et al., 1990; Lindvall et al., 1990; Sanberg et al., 1994; Bjorklund and Stenevi, 1985; Freeman et al., 1994). In some cases, transplanted neural tissue can survive and form connections with the CNS of the recipient, i.e. the host (Wictorin et al., 1990). When successfully accepted by the host, the transplanted cells and/or tissue (i.e.. the graft) have been shown to ameliorate the behavioral deficits associated with the disorder (Sanberg et al., 1994). The obligatory step for the success of this kind of treatment is to have enough viable cells available for the transplant.
Currently, fetal neural tissue is the primary graft source for neural transplantation (Lindvall et al., 1990; Bjorklund, 1992; Isacson et al., 1986; Sanberg et al., 1994). Other viable graft sources include adrenal chromaffin cells and various cell types that secrete neural growth factors and trophic factors. The field of neural tissue transplantation as a productive treatment protocol for neurodegenerative disorders has received much attention resulting in its progression to clinical trials. Preliminary results and clinical observations are promising but obtaining enough viable cells remains a problem.
For example, one treatment for Parkinson's disease (PD) intracerebral transplantation therapy, has accentuated research interest in restoring some of the circuits in the nigrostriatal pathway Lindvall et al., 1990; Lindvall, 1994; Freeman et al., 1995; Kordower et al., 1995, 1996!. While the initial findings are encouraging and have resulted in behavioral improvements in patients with PD, the current clinical protocols for intracerebral transplantation have to be improved in terms of increasing short- and long-term survival of embryonic dopaminergic (DA) cells, and to find alternative graft sources to avoid the problem with lack of donor tissue obtained from elective abortions.
Lately, research has focused on finding trophic factors, able to increase the survival of DA cells prepared for transplantation, maintain the in situ survival post-transplantation of embryonic DA neurons transplanted into the striatum, as well as increase graft volume, and thereby re-innervate a larger part of the caudate and putamen which has been shown to have effect both in vitro and in vivo. Trophic factors such as NGF, bFGF, EGF, IGF I and II, TGF.beta.1-3, PDGF, BDNF, and GDNF Engele and Bohn, 1996; Mayer et al., 1993; Knusel et al., 1990, 1991; Poulsen et al., 1996; Nikkhah et al., 1993; Othberg et al., 1995; Hyman et al., 1991, 1993; Lin et al., 1993! have been investigated and shown to have pronounced effects in vitro, however the effects in vivo have yet to be further established. Reversal of MPTP and 6-OHDA lesions in primates Gash et al., 1996! and rats, as well as increased graft survival, have been demonstrated by the addition of NGF or bFGF to the cell suspension prior to grafting Chen et al., 1996; Dunnett et al., 1995!, or by transplanting neurons derived from a neural progenitor (CINP) cell line, transduced retrovirally with NGF Martinez-Serrano et al., 1995! and astrocytes transduced with BDNF Yoshimoto et al., 1996!. GDNF has been shown to increase graft survival, extend fiber outgrowth and alleviate behavioral effects after 6-hydroxydopamine lesions in the striatum of rats Sauer et al., 1994; Johansson et al., 1995; Bowenkamp et al., 1995; Rosenblad et al., 1996; Olson, 1996!.
In treating disease it is often useful to treat tissue locally, rather than systemically, with trophic factors, particularly areas of tissue damage as for example in wound healing. Additionally, it is becoming increasingly recognized that multiple trophic factors acting in concert are likely to be necessary for successful treatment. Further, the availability of multiple trophic factors at various time points during treatment may be necessary to enhance successful treatment.
Long term maintenance of functional recovery has been observed in a diabetic animal model utilizing a novel transplantation treatment protocol utilizing isolated islet cells and Sertoli cells. It is clear that the efficacy of the treatment is due to the presence of the Sertoli cells, in part, due to their known immunosuppressive secretory factor. (Selawry and Cameron, 1993; Cameron et al., 1990). However, Sertoli cells are also known to secrete a number of important trophic growth factors.
Accordingly, it would be desirable to utilize Sertoli cells as a source for trophic factors to improve viability and growth of cells/tissues for transplantation, cryopreservation and for trophic factor support of damaged tissue. Sertoli cells actively participate in the genesis of spermatozoa. The Sertoli cells make a wide variety of nutritive, trophic and regulatory proteins, amongst them a wide variety of trophic factors and their receptors Skinner, 1993!. Individual trophic factors, as listed in Table 1, have been evaluated, but biological requirements are complex and the interaction of various components often necessary to effectively provide the necessary stimulants. It would be useful to design preparations that provide the various components and interactions to effectively improve viability, maturation, number and growth of cells/tissues for transplantation and for wound healing and cryopreservation.
Cell transplantation therapies are optimized by the availability of cryopreserved cells which have high viability. Transplantable cells, such as fetal brain cells, do not withstand cryopreservation well. Therefore, it would be desirable to have a method for enhancing the preservation and viability of cryopreserved cells in order to optimize the function of the cells and to obtain the resultant benefits to the transplant recipient.